Autophagy contributes to degradation of Hirano bodies

Hirano bodies are actin-rich inclusions reported most frequently in the hippocampus in association with a variety of conditions including neurodegenerative diseases, and aging. We have developed a model system for formation of Hirano bodies in Dictyostelium and cultured mammalian cells to permit detailed studies of the dynamics of these structures in living cells. Model Hirano bodies are frequently observed in membrane-enclosed vesicles in mammalian cells consistent with a role of autophagy in the degradation of these structures. Clearance of Hirano bodies by an exocytotic process is supported by images from electron microscopy showing extracellular release of Hirano bodies, and observation of Hirano bodies in the culture medium of Dictyostelium and mammalian cells. An autophagosome marker protein Atg8-GFP, was co-localized with model Hirano bodies in wild type Dictyostelium cells, but not in atg5(-) or atg1-1 autophagy mutant strains. Induction of model Hirano bodies in Dictyostelium with a high level expression of 34 kDa DeltaEF1 from the inducible discoidin promoter resulted in larger Hirano bodies and a cessation of cell doubling. The degradation of model Hirano bodies still occurred rapidly in autophagy mutant (atg5(-)) Dictyostelium, suggesting that other mechanisms such as the ubiquitin-mediated proteasome pathway could contribute to the degradation of Hirano bodies. Chemical inhibition of the proteasome pathway with lactacystin, significantly decreased the turnover of Hirano bodies in Dictyostelium providing direct evidence that autophagy and the proteasome can both contribute to degradation of Hirano bodies. Short term treatment of mammalian cells with either lactacystin or 3-methyl adenine results in higher levels of Hirano bodies and a lower level of viable cells in the cultures, supporting the conclusion that both autophagy and the proteasome contribute to degradation of Hirano bodies.     

Multiplexed Biomolecular Detection Based on Single Nanoparticles Immobilized on Pneumatically Controlled Microfluidic Chip

Plasmonics - A microfluidic chip integrated with pneumatically controlled valves was developed for multiplexed biomolecular detection via localized surface plasmonic resonance (LSPR) of single gold...     

Mechanisms underlying vernalization-mediated VIN3 induction in Arabidopsis

VERNALIZATION INSENSITIVE 3 (VIN3) is required for vernalization-mediated repression of FLOWERING LOCUS C (FLC) in Arabidopsis. The induction of VIN3 by long-term exposure to cold is one of earliest events in vernalization response. However, molecular mechanisms underlying for the VIN3 induction are poorly understood. Recently, we reported that the constitutive repression of VIN3 in the absence of the cold exposure is due to multiple repressive chromatin modifying components, including a transposable element (TE)-derived sequence, LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1) and POLYCOMB REPRESSION COMPLEX 2 (PRC2). In addition, the maximum level of VIN3 induction requires EARLY FLOWERING 7 (ELF7) and EARLY FLOWERING IN SHORDAYS (EFS), which are components of activating chromatin modifying complexes. Furthermore, dynamic changes in histone modifications at VIN3 chromatin are observed during the course of vernalization. Thus, mechanisms underlying the induction of VIN3 include changes at the level of chromatin.Key words: vernalization, flowering, chromatinVernalization can be defined as “the acquisition or acceleration of the ability to flower by a chilling treatment.”¹ To maximize floral reproductive capability, plants in temperate climates have evolved a vernalization requirement to prevent flowering before the winter season and to ensure flowering in the spring. Vernalization response provides plants with competence to flower, rather than to induce flowering itself, through changes that remain stable even after cold exposure. This process is an epigenetic switch, whereby molecular changes remain stable throughout subsequent mitotic divisions despite the absence of initiating stimulus, cold exposure.^2–4 Vernalization response can be thought of as being comprised of two phases. The first is a cold perception system that measures the cumulative time of exposure to cold. The second phase is essentially the output of cold perception system: when a sufficient duration of cold has been perceived, a series of changes of gene expression ensue, ultimately leading to the epigenetic repression of FLC.     

A New Species of Theridion Walckenaer (Araneae: Theridiidae) from Korea

A new species, Theridion longipili sp. nov. (Theridiidae), is described from Korea. It is easily distinguished from other congeners by the structure of conductor, radix, median apophysis, and the female epigynum and internal genitalia.     

Peptide-based polyclonal antibody against mosquito 14-3-3ζ recognizes 14-3-3 homolog from dipteran and lepidopteran insects

The 14-3-3 proteins are known to play an important regulatory role in apoptosis, and various cell signaling cascades. However, no investigation on mosquito 14-3-3 has been reported. To investigate the role of 14-3-3 proteins in mosquito midgut cells undergoing apoptosis, we decided to take advantage of Anopheles gambiae genome data, and were able to find Ag14-3-3ζ cDNA and protein sequences from Ensembl ( http://www.ensembl.org ). Further in silico analysis using BLAST search revealed that Ag14-3-3ζ protein is a polypeptide of 248 amino acids, and shares high identity with 14-3-3ζ homologues from Aedes aegypti (100%), Drosophila melanogaster (96%) and Bombyx mori (93%). Due to the perfect match and high homology, we hypothesized that Ag14-3-3ζ peptide antibody may recognize 14-3-3ζ homologs from other anopheline mosquitoes and insects. We thus generated 14-3-3ζ polyclonal antibody against a unique region located in the C-terminal end of Ag14-3-3ζ after in silico epitope analysis. As expected, zoo-western blot analysis of 14-3-3 proteins revealed that a polyclonal antibody against Ag14-3-3ζ peptide recognizes 14-3-3 homologs from dipteran and lepidopteran insects. To our knowledge, this is the first report on polyclonal antibody production against mosquito 14-3-3ζ. The mosquito-based 14-3-3ζ antibody will be very useful for studying the functional characterization of 14-3-3ζ in the context of host–pathogen interactions in midgut and other immune cells.     

Facile fabrication of distance-tunable Au-nanorod chips for single-nanoparticle plasmonic biosensors

In this work, we present a simple and effective method to fabricate distance-controllable, Au nanorod (AuNR) chips thorough electrostatic assembly. Cetyltrimethylammonium bromide (CTAB)-capped AuNRs were immobilized on a hydroxyl-functionalized glass substrate by immersion of the glass into AuNR-suspension. The electrostatic surfacial assembly of AuNRs offers significant advantages over conventional thiol-induced chemistry, i.e., direct control of self-assembly of AuNRs, easy fabrication in ambient environment and most importantly, broad range of tunable inter-particle distance, ranging from 0.25 to 10 μm. The mechanism of time-dependant deposition process of AuNRs was described via competitive bindings of AuNRs and free CTAB molecules in AuNR-suspension. In addition, the electrostatically anchored AuNRs on a glass substrate provide sufficient stability under harsh experimental conditions with flow of basic/acidic solutions and organic solvents with different polarity. The feasibility of the AuNR-chips fabricated by the proposed method for single-nanoparticle plasmonic biosensors was demonstrated by the plasmonic measurement of aptamer-thrombin binding event. The corresponding limit of detection of thrombin molecule was found to be ～278 pM based on the signal to noise ratio of 4.     

LSPR biomolecular assay with high sensitivity induced by aptamer-antigen-antibody sandwich complex

Herein we demonstrate a sensitive approach for protein detection based on peak shifts of localized surface plasmon resonance (LSPR) induced by aptamer-antigen-antibody sandwich structures. The applicability of the proposed method is demonstrated using human α-thrombin as a model analyte. While the binding of thrombin to its specific receptor, thrombin binding aptamer (TBA) modified on Au nanorods (AuNRs), causes a measurable LSPR shift, a subsequent binding of an anti-thrombin antibody to the captured thrombin can exhibit a nearly 150% amplification in the LSPR response. This enhanced signal essentially leads to an improvement of limit of detection (LOD) by more than one order of magnitude. In addition, the use of TBA as thrombin recognition units makes the biosensor reusable. The feasibility of the proposed method was further exploited by the detection of thrombin in human serum, opening the possibility of a real application for diagnostics and medical investigations.     

Simultaneous quantification of cyanobacteria and Microcystis spp. using real-time PCR

In order to develop a protocol to quantify cyanobacteria and Microcystis simultaneously, the primers and probe were designed from the conserved regions of 16S rRNA gene sequences of cyanobacteria and Microcystis, respectively. Probe match analysis of the Ribosomal Database Project showed that the primers matched with over 97% of cyanobacterial 16S rRNA genes, indicating these can be used to amplify cyanobacteria specifically. The TaqMan probe, which is located between two primers, matched with 98.2% of sequences in genus GpXI, in which most Microcystis strains are included. The numbers of cyanobacterial genes were estimated with the emission of SYBR Green from the amplicons with two primers, whereas those of Microcystis spp. were measured from the fluorescence of CAL Fluor Gold 540 emitted by exonuclease activity of Taq DNA polymerase in amplification. It is expected that this method enhances the accuracy and reduces the time to count cyanobacteria and potential toxigenic Microcystis spp. in aquatic environmental samples.